At the present state of analysis the central part of the kappa locus comprises four contigs of together 1.2 Mb and contains 55 Vkappa genes. It is flanked by the 3' part of the locus with 22 Vkappa genes in 0.4 Mb (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) and the 5' part with 63 Vkappa genes in six contigs of together 1.5 Mb (F. Röschenthaler et al., accompanying report). The 5' and the central regions have one large contig in common. A part of the central region is linked to the 3' region resulting in a 1.1-Mb contig. The structure of the contigs was established mainly by the analysis of overlapping cosmid clones derived from genomic DNA and yeast and bacterial artificial chromosomes (YACs and BACs) and by PCR techniques. Pulsed-field gel electrophoresis of YAC digests indicated that three gaps between the contigs of the central region are 10-40 kb in size, comprising together about 90 kb. Internal duplications in this part of the locus and rearranged YACs were the major problems of the structural work. Structural details are to be found on the Internet at http://www.med.uni-muenchen.de/biochemie/zach au/kappa.htm. In a concluding section of the report the mouse kappa locus is compared to the human one and some aspects of the evolution of the kappa locus are discussed.
The central regions of the kappa locus, the so-called A regions, have been fully characterized on cosmid and phage lambda clones. The regions, which are parts of the C kappa-proximal and -distal copies of the locus and are, therefore, called Ap and Ad regions, comprise about 140 kb each and contain together 30 V kappa genes and pseudogenes. The A regions have been linked on their 5' sides to the O regions and on their 3' sides to the L regions. Chromosomal walking has eliminated a previous gap in the Ap region. Detailed restriction maps of the Ap and Ad regions and the sequences of 9 V kappa genes are reported. Four events, which have occurred in evolution probably after the duplication of the A region, were identified: the insertion of an Alu element in Ad; the insertion of part of a LINE element in Ap; the deletion of a 17.5-kb fragment including one V kappa gene from Ap; the sequence divergence of duplicated V kappa gene regions which ranges among the five pairs studied here from 0 to 14 bp per kb and converted two genes to pseudogenes while their duplicates stayed functional. An analysis of the A regions of the lymphoid cell lines RPMI 6140 and GM607 confirmed the previous finding that the V kappa-J kappa rearrangement in these cell lines had occurred by deletion and inversion mechanisms, respectively. Thus, the structural data contribute to the understanding of the evolution and the functioning of the A regions of the kappa locus.
The L regions are parts of the C kappa proximal (p) and distal (d) copies of the human immunoglobulin kappa locus and are therefore called the Lp and Ld regions. The two regions with their 25 V kappa genes and pseudogenes have now been cloned, thus completing the cloning of the kappa locus. Lp has been linked to the neighboring Ap and B regions, while Ld was linked to Ad. There is good evidence that at the other side of Ld, i.e. towards the centromere, the end of the locus has been reached. Most of the cloning and linking was achieved by chromosomal walking, employing cosmid and phage lambda clones. No such clones could be found for three small gaps. Two of them were closed by a polymerase chain reaction strategy; the third one was characterized by genomic blot hybridization experiments and eventually bridged by a yeast artificial chromosome clone. Early in evolution, a stretch of about 25 kb which comprised three V kappa genes near the 5' end of the L region precursor must have been duplicated, such that the later duplication of large parts of the kappa locus resulted in the appearance of two very similar three-gene regions in each, Lp and Ld. Two deletions in the central parts of the L regions, on the other hand, must have occurred after the duplication of the locus, since they are found in Lp and Ld in different positions.
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